Development of biophysical approaches to investigate high-resolution structure and dynamics of membrane proteins

NIH Pandemic-Era Grants

Pandemic Era Grants

2024

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Principal Investigator: Ayyalusamy  Ramamoorthy
Organization: FLORIDA STATE UNIVERSITY
Fiscal Year: 2024
Award: $396,250
Funding agency: National Institute of General Medical Sciences

Abstract
Functional reconstitution of membrane proteins has been the major roadblock for the application of NMR and
other biophysical techniques to investigate their high-resolution dynamic structures in a native membrane
environment. In this application, we propose to develop approaches to enable high-resolution structural studies
of membrane proteins and protein-protein complexes by a variety of biophysical techniques. We will develop
nanodisc technology for detergent-free direct extraction and functional reconstitution of membrane proteins for
structural studies of a variety of membrane proteins including single-pass transmembrane proteins (such as
mammalian cytochromes and heme oxygenase) and integral membrane proteins (including GPCRs and
Guanidine exporter). Synthetic polymers developed in our laboratory exhibit the ability to form nanodiscs with
easily controllable sizes (from ~8 to ~60 nm diameter), are stable against pH and divalent metal ions and capable
of directly extracting membrane proteins. Our preliminary results demonstrate that these nanodiscs (<20 nm
diameter) and macro-nanodiscs (>20 nm diameter) represent an exciting system for solution and solid-state
NMR studies of membrane proteins.
 We also propose to use the newly developed nanodisc technology and NMR approaches to investigate
the structural interactions of mammalian cytochrome-P450 (P450) with its redox partners (P450-reductase (CPR)
and cytochrome-b5 (b5)) to better understand how redox partners regulate P450 catalysis and how P450s
metabolize chemically diverse substrates. The structural aspects pertaining to the catalytic activity of P450s
continue to remain elusive due to a lack of high-resolution structures in their full-length, active forms. Presently,
structural studies of P450s are restricted to various truncated mammalian and water-soluble bacterial P450
homologs. In this study, we will investigate the structure, dynamics and transmembrane domain orientation of
full-length mammalian P450s (2B4, 3A4 and 3A5 isoforms) alone and in complex with its redox partner b5 and
CPR, incorporated in nanodiscs, using a combination of high-resolution solution and solid-state NMR techniques.
We will also investigate the ternary P450-b5-CPR complex in nanodiscs in the presence of substrates to
elucidate the molecular origin of the strikingly different effects CPR and b5 have on P450 2B4 catalysis. The
outcome of the proposed studies on P450-redox complexes will provide structure and dynamics/function
principles regulating P450 metabolism of a wide variety of substrates. The results obtained from this study will
also be useful to design potent drugs to ultimately treat and prevent diseases including cancer.

Terms: <Breast Cancer><Cancers><Catalysis><Cell Membrane Lipids><Chemicals><Complex><Cytochrome P-450><Cytochrome P-450 Enzyme System><Cytochrome P450><Cytochrome P450 Family Gene><Cytochromes><Dehydrogenases><Detergents><Development><Diameter><Disease><Disorder><Drugs><Environment><Exhibits><Ferroprotoporphyrin><G Protein-Complex Receptor><G Protein-Coupled Receptor Genes><G-Protein-Coupled Receptors><GPCR><GeneHomolog><Guanidines><Heme><Homolog><Homologous Gene><Homologue><Human><Hydrogen Oxide><Integral Membrane Protein><Intermediary Metabolism><Intrinsic Membrane Protein><Ions><Isoforms><Laboratories><Length><Malignant Breast Neoplasm><Malignant Neoplasms><Malignant Tumor><Malignant Tumor of the Prostate><Malignant neoplasm of prostate><Malignant prostatic tumor><Medication><Membrane><Membrane Lipids><Membrane Protein Gene><Membrane Proteins><Membrane-Associated Proteins><Metabolic Processes><Metabolism><Modern Man><Molecular><Outcome><Oxidation-Reduction><Oxidoreductase><Oxidoreductase Gene><Oxygenases><P450><Pharmaceutical Agent><Pharmaceutical Preparations><Pharmaceuticals><Pharmacologic Substance><Pharmacological Substance><Polymers><Prostate CA><Prostate Cancer><Prostate malignancy><Prostatic Cancer><Protein Isoforms><Proteins><Protoheme><Redox><Reductases><Resolution><SSNMR><Structure><Surface Proteins><System><TM Domain><Techniques><Transmembrane Domain><Transmembrane Protein><Transmembrane Protein Gene><Transmembrane Region><Water><biophysical approaches><biophysical methodology><biophysical methods><biophysical techniques><design><designing><developmental><divalent metal><drug/agent><ferroheme><insight><malignancy><malignant breast tumor><membrane reconstitution><membrane structure><nanodisc technology><nanodisk><neoplasm/cancer><oxidation reduction reaction><pharmaceutical><polymer><polymeric><prevent><preventing><protein complex><resolutions><solid state NMR><solid state nuclear magnetic resonance>